Biological organisms such as stem cells, tissues, including, but not limited to, immature oocytes, gametes, zygotes, embryos, cleavage stage embryos, blastocyst stage embryos, and the like, are commonly cultured, grown, and may be preserved by freezing and/or vitrification. Petri dishes are generally designed for use by the individual technician for use in a broad range of procedures and applications. Currently there is no Petri dish or similar device that is specifically designed for use during the preparation of the specimens for specimen culture and freezing and/or for thawing the specimens. More specifically, there is no dish or other device that is designed to be used for the multi step preparation of human or animal specimens in the specimen culturing and cryo-preservation and thawing processes.
Currently, there are two methods used for the cryo-preservation of the aforesaid types of specimens, the first of which is referred to as conventional cryo-preservation, and second of which is referred to as vitrification.
The conventional cryo-preservation method refers to the forming of ice crystals during the freezing process and the removal of such ice crystals during the thawing process.
The conventional vitrification method refers to the solidification of the specimens in a solution of reduced and lowered temperature, not by ice crystallization formation, but by extreme elevation of sample viscosity during cooling. Then the specimens are warmed as opposed to thawed. The amounts of specific materials, such as cryo-preservatives and culture medium may vary between the methods.
We will refer herein to these two different methods as being interchangeable, although they may vary in the number of steps, materials and terminologies used when performing certain different procedures or protocols. Therefore, for the purpose of describing procedures, steps or protocols herein, we will refer to “freeze” or “vitrification” when the specimen is being prepared for either of the procedures of cryopreservation, or being prepared for storage, and “thaw” when the specimen is subjected to warming or being taken out of the cryopreservation state.
The problems posed by the current methodology is that technicians are relying on common Petri dishes in these types of specimen procedures in the preparation of human and animal specimens for cryo-preservation and thawing. These dishes are not well suited for these procedures since they may not contain a desired number of wells, are not configured to facilitate these types of processes, and do not allow the technician to readily a fixed set of protocols or procedures each time.
Currently, common Petri dishes used to freeze and thaw the specimens may have flat bottoms, may be with or without molded integral multiple wells, and most will not contain the number of wells needed to complete the desired tasks. For those dishes without wells, the technician may simply create a droplet or a micro-droplet of the solution to be used and then put the specimen into those drops. When the dishes have wells, they may have a single well or up to four wells.
The use of micro-droplets also extends the setup time (which may be up to one hour), the time for performing the steps and procedures, and also may put the specimens at risk, by the possibility of the droplets collapsing thereby exposing the specimens to oil, air, and permanent damage. The specimens must also be handled a number of times which puts the specimens at risk of damage.
Additional problems faced by use of these common Petri dishes is that the procedures in question utilize several different media solutions and the technician must use dishes that may not adapt well to the task, or the technician may try to adapt his or her procedures, steps or protocols, and the timing of the steps, to comply with the restrictions imposed by the dishes used. The result is an ineffective performance of the procedures to be followed and thus poor results. The reduced number of wells may also force the individual technicians to use more than one dish adding to the confusion, creating possible errors, and complicating the performance of the procedures.
In these procedures, the use of multiple vials and different types of medium solutions within these vials, which may contain different concentrations of ingredients or greater of lesser amounts of a particular ingredient, will occur.
An example of the above can be where Vial #1 is a washing solution, and is used in the beginning of preparation of human oocytes. The washing solution is a base media and does not contain any cryo-preservatives; a Vial #2 contains the same base media along with 1,2-propanediol (PrOH), ethylene glycol, glycol, as a cryo-preservative; and a Vial #3 contains the same media as Vial #2 plus a small amount of sucrose.
The solutions used for the cryo-preservation procedures may also contain the ingredients in various levels and may use up to five different vials. An example of this is: Vial #1 may contain 100% base media; Vial #2 may contain 90% media and 10% PrOH; Vial #3 may contain 80% media and 20% PrOH; Vial #4 may contain 70% media and 30% PrOH; and Vial #5 may contain 60% media and 40% PrOH. Thus each of the successive step vials contains a solution in which a specimen is to be immersed, with each of the solutions containing progressively less media and progressively more of the cryo-protectant. The successive specimen treatment steps are timed steps, with the time specimen immersion time for each step being from five to ten minutes, depending on the established protocol in use. Certain protocols in thawing currently use seven different solutions in seven separate vials. These protocols take at least thirty five minutes to complete. The specimens are transferred from one solution to the next during the protocol. Transferring the specimens puts them at risk of damage. Thus, the present protocols which require transferring the specimens from one solution to another are labor intensive, equipment intensive, and present a risk of damage to the specimens during the treatment protocol.
The above-noted protocols for specimen treatment also require that the media and cryo-protectant solutions be overlain in the dish wells with a mineral or paraffin oil to prevent the solutions from evaporating during the treatment steps. The mineral or paraffin oil covers complicate the procedure since the technician must not pick up the oil in the micro tools used to transfer the specimens from one solution to the next. Thus, in order to perform the present protocols, the vials must be placed in the proper order or sequence, to properly prepare the specimens for cryo-preservation or thawing.
Current dishes require the technician to move the specimens from one solution to the next by the use of micro tools and microscopes to find and move the specimens. This is very time and equipment consuming, and the multiple handling of the specimens can also contribute to the problem of growing and survival of the specimens during the protocol in question.
Currently, researchers in stem cell work utilize a series of media solutions, adding ingredients or additional media at specified time intervals and may at times add supplements such as growth hormones and proteins. The cells being grown remain in the desired dish or apparatus for several days, or even for up to two-three weeks. In this procedure, it would be desirable to add additional medium ingredients or additives without having to move the cells, and to be able to change the overall concentrations of certain ingredients by adding ingredients at various times.
It would be desirable to provide an apparatus and method for performing multi-step protocols for preparing specimens for cryo-preservation and/or thawing which does not require multiple vials for containing varying solution concentrations, and which does not require physically transferring specimens being treated in the protocol from one solution vial to another.
It would be highly desirable to provide an apparatus which will promote the growth of stem cells or cells and the changing of the ingredient concentrations without moving the specimens and by adding or withdrawing medium and other components.